Methods and compositions for assaying homocysteine

ABSTRACT

This invention relates generally to the field of homocysteine detection. In particular, the invention provides a method for determining homocysteine presence or concentration in samples, which method comprises: contacting a sample containing or suspected of containing Hcy with a Hcy co-substrate and a Hcy converting enzyme in a Hcy conversion reaction to form a Hcy conversion product and a Hcy co-substrate conversion product; and assessing the Hcy co-substrate conversion product to determine the presence, absence and/or amount of the Hcy in the sample. A kit for assaying homocysteine based on the same principle is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of provisional applicationU.S. Ser. No. 60/486,865, filed Jul. 10, 2003, the contents of which areincorporated by reference in their entirety.

TECHNICAL FIELD

This invention relates generally to the field of homocysteine detection.In particular, the invention provides a method for determininghomocysteine (Hcy) presence or concentration in samples in whichhomocysteine reacts with a Hcy co-substrate in a Hcy conversion reactioncatalyzed by a Hcy converting enzyme to form a Hcy conversion productand a Hcy co-substrate conversion product, and the Hcy co-substrateconversion product is assessed to determine the presence and/orconcentration of the Hcy in the sample. A kit for assaying homocysteinebased on the same principle is also provided.

BACKGROUND OF THE INVENTION

Total concentration of homocysteine in body fluids, such as plasma orserum, is an important marker for disease. For example, homocysteinequantification can be an important risk indicator for cardiovasculardisease, can be a sensitive marker of cobalamin and folate deficiencies,and can be used to diagnose in-born errors in metabolism known ashomocystinuria. Homocysteine quantification has also been reported asuseful in assessing birth defects in pregnant women and cognitiveimpairment in the elderly. See Frantzen, et al., Enzyme ConversionImmunoassay for Determining Total Homocysteine in Plasma or Serum,Clinical Chemistry 44:2, 311-316 (1998).

Homocysteine (Hcy) is a thiol-containing amino acid formed frommethionine during S-adenosylmethionine-dependent transmethylationreactions. Intracellular Hcy is remethylated to methionine, or isirreversibly catabolized in a series of reactions to form cysteine.Intracellular Hcy is exported into extracellular fluids such as bloodand urine, and circulates mostly in oxidized form, and mainly bound toplasma protein (Refsum, et al., Annu. Rev. Medicine, 49:31-62 (1998)).The amount of Hcy in plasma and urine reflects the balance between Hcyproduction and utilization. This balance may be perturbed by clinicalstates characterized by genetic disorders of enzymes involved in Hcytranssulfuration and remethylation (e.g., cystathionine β-synthase andN^(5,10)-methylenetetrahydrofolate reductase or dietary deficiency ofvitamins (e.g., vitamin B₆, B₁₂ and folate) involved in Hcy metabolism(Baual, et al., Cleveland Clinic Journal of Medicine, 64:543-549(1997)). In addition, plasma Hcy levels may also be perturbed by somemedications such as anti-folate drugs (e.g., methotrexate) used fortreatments of cancer or arthritis (Foody, et al., Clinician Reviews,8:203-210 (1998))

Severe cases of homocysteinemia are caused by homozygous defects ingenes encoding for enzymes involved in Hcy metabolisms. In such cases, adefect in an enzyme involved in either Hcy remethylation ortranssulfuration leads to as much as 50-fold elevations of Hcy in theblood and urine. The classic form of such a disorder, congenitalhomocysteinemia (Hcyemia), is caused by homozygous defects in the geneencoding cystathionine β-synthase (CBS). These individuals suffer fromthromboembolic complications at an early age, which result in stroke,myocardial infarction, renovascular hypertension, intermittentclaudication, mesenteric ischemic, and pulmonary embolism. Such patientsmay also exhibit mental retardation and other abnormalities resemblingectopia lentis and skeletal deformities (Perry T., Homocysteine:Selected aspects in Nyham W. L. ed. Heritable disorders of amino acidmetabolism. New York, John Wiley & Sons, pp. 419-451 (1974)). It is alsoknown that elevated Hcy levels in pregnant women is related to birthdefects of children with neurotube closures (Scott, et al., “Theetiology of neural tube defects” in Graham, I., Refsum, H., Rosenberg,I. H., and Ureland P. M ed. “Homocysteine metabolism: from basic scienceto clinical medicine” Kluwer Academic Publishers, Boston, pp. 133-136(1995)). Thus, the diagnostic utility of Hcy determinations has beenwell documented in these clinical conditions.

It has been demonstrated that even mild or moderately elevated levels ofHcy also increase the risk of atherosclerosis of the coronary, cerebraland peripheral arteries and cardiovascular disease (Boushey, et al.,JAMA, 274:1049-1057 (1995)). The prevalence of Hcyemia was shown to be42%, 28%, and 30% among patients with cerebral vascular disease,peripheral vascular disease and cardiovascular disease, respectively(Moghadasian, et al., Arch. Intern. Med., 157:2299-2307 (1997)). Ameta-analysis of 27 clinical studies calculated that each increase of 5μM in Hcy level increases the risk for coronary artery disease by 60% inmen and by 80% in women, which is equivalent to an increase of 20mg/dl⁻¹ (0.5 mmol/dl⁻¹) in plasma cholesterol, suggesting that Hcy, as arisk factor, is as strong as cholesterol in the general population.Results from these clinical studies concluded that hyperhomocysteinemiais an emerging new independent risk factor for cardiovascular disease,and may be accountable for half of all cardiovascular patients who donot have any of the established cardiovascular risk factors (e.g.,hypertension, hypercholesterolemia, cigarette smoking, diabetesmellitus, marked obesity and physical inactivity).

Mild homocysteinemia is mainly caused by heterozygosity of enzymedefects. A common polymorphism in the gene for methylenetetrahydrofolatereductase appears to influence the sensitivity of homocysteine levels tofolic acid deficiency (Boers, et al., J. Inher. Metab. Dis., 20:301-306(1997)). Moreover, plasma homocysteine levels are also significantlyincreased in heart and renal transplant patients (Ueland, et al., J.Lab. Clin. Med, 114:473-501 (1989)), Alzheimer patients (Jacobsen, etal., Clin. Chem., 44:2238-2239 (1998)), as well as in patients ofnon-insulin-dependent diabetes mellitus (Ducloux, et al., Nephrol. Dial.Transplantl, 13:2890-2893 (1998)). The accumulating evidence linkingelevated homocysteine with cardiovascular disease has prompted theinitiation of double-blind, randomized and placebo controlledmulticenter clinical trials to demonstrate the efficacy of loweringplasma Hcy in preventing or halting the progress of vascular disease(Diaz-Arrastia, et al., Arch. Neurol., 55:1407-1408 (1998)).Determination of plasma homocysteine levels should be a common clinicalpractice.

As a risk factor for cardiovascular disease, the determination of totalplasma Hcy levels (reduced, oxidized and protein-bound) has beenrecommended in clinical setting (Hornberger, et al., American J. ofPublic Health, 88:61-67 (1998)). Since 1982, several methods fordetermining total plasma Hcy have been described (Mansoor, et al., Anal.BioChem., 200:218-229 (1992); Steir, et al., Arch. Intern. Med.,158:1301-1306 (1998); Ueland, et al., Clin. Chem., 39:1764-1779 01993);and Ueland, et al., “Plasma homocysteine and cardiovascular disease” inFrancis, R. B. Jr. eds. Atherosclerotic Cardiovascular Disease,Hemostasis, and Endothelial Function. New York, Marcel Dokker,pp.183-236 (1992); see, also, Ueland, et al., “Plasma homocysteine andcardiovascular disease” in Francis, R. B. Jr. eds. AtheroscleroticCardiovascular Disease, Hemostasis, and Endothelial Function. New York,Marcel Dokker, pp. 183-236 (1992)). The assay of total Hcy in plasma orserum is complicated by the fact that 70% of plasma Hcy is protein-boundand 20-30% exists as free symmetric or mostly asymmetric mixeddisulfides. Free reduced Hcy exists in only trace amounts (Stehouwer, etal., Kidney International, 55308-314 (1999)).

Most of the methods require sophisticated chromatographic techniquessuch as HPLC, capillary gas chromatography, or mass spectrometry (GC/MS)to directly or indirectly (e.g., enzymatic conversion of Hcy to SAH(S-adenosylhomocysteine) by SAH hydrolase followed by HPLC or TLCseparation) measure Hcy. Radioenzymatic conversion of Hcy toradiolabeled SAH by SAH hydrolase prior to TLC separation has also beenused. In these assays, chromatographic separation, which is oftentime-consuming and cumbersome to perform, is a common key step of thesemethods. More particularly, these methods require highly specialized andsophisticated equipment and well-trained analytic specialists. The useof such equipment is generally not well-accepted in routine clinicallaboratory practice.

Immunoassays for Hcy that use a monoclonal antibody against SAH (Araki,et al., J. Chromatog., 422:43-52 (1987)) are also known. These assaysare based upon conversion of Hcy to SAH, which is then detected by amonoclonal antibody. Monoclonal antibody against albumin-bound Hcy hasbeen developed for determination of albumin-bound Hcy (Stabler, et al.,J. Clin. Invest., 81:466-474 (1988)), which is the major fraction oftotal plasma Hcy. Other immunological protocols are also available (see,e.g., U.S. Pat. Nos. 5,631,127, 5,827,645, 5,958,717, 6,063,581 and5,885,767). Though immunoassays avoid a time-consuming chromatographicseparation step and are amenable to automation, production of monoclonalantibody is expensive, somewhat unpredictable, and often requiressecondary or even tertiary antibodies for detection. Recently, enzymaticmethods for homocysteine assay have been reported (Matsuyama, et al.,Clinical Chemistry, 47:2155-2156 (2001); Tan et al., Clinical Chemistry,49:1029-1030 (2003); U.S. Pat. Nos. 5,885,767, 5,998,191, 6,046,017,6,174,696, 6,436,658 and 6,664,073 B1), all of these describehomocysteine assays based on the assessment of homocysteine conversionproducts generated by homocysteine converting enzymes.

Other methods for determining homocyteine in a sample are described inU.S. Pat. No. 6,686,172 and U.S. Pat. App. Pub. No. 2002/0119507.

An efficient and accurate assay, that can be carried out withoutnecessity for highly skilled personnel or complex analytical chemistryequipment, has been needed. The present invention addresses the aboveand other related concerns in the art.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to an assay forhomocysteine in a sample. According to this assay, a sample containingor suspected of containing homocysteine (Hcy) is contacted with a Hcyco-substrate and a Hcy converting enzyme in a Hcy conversion reaction toform a Hcy conversion product and a Hcy co-substrate conversion product;and the Hcy co-substrate conversion product is assessed to determine thepresence, absence and/or amount of the Hcy in said sample.

In some embodiments of the invention, the Hcy co-substrate conversionproduct is assessed without chromatographic separation.

In some embodiments, the Hcy co-substrate is S-adenosylmethionine (SAM),the Hcy converting enzyme is S-adenosylmethionine (SAM)-dependenthomocysteine S-methyltransferase, the Hcy conversion product ismethionine (Met) and the Hcy co-substrate conversion product isS-adenosyl-L-homocysteine (SAH), and the SAH is assessed to determinethe presence, absence and/or amount of the Hcy in the sample.

The SAM can be used in any suitable form. For example, the SAM is addedto the sample directly. In another example, the SAM is produced by afurther reaction, e.g., produced from ATP and Met by a SAM synthase.

The SAH may be converted to Hcy and adenosine (Ado), and Ado is assessedto determine the presence, absence and/or amount of the Hcy in thesample. In some embodiments, the SAH is contacted with a SAH hydrolaseto generate Hcy from SAM, which is cycled into the Hcy conversionreaction by the SAM-dependent homocysteine S-methyltransferase to form aHcy co-substrate based enzyme cycling reaction system, and adenosine(Ado), which is assessed to determine the presence, absence and/oramount of the Hcy in the sample.

The Ado may be assessed by any suitable methods known in the art such asimmunological or enzymatic methods. The Ado may be assessed directly orindirectly. For example, the Ado may be assessed indirectly by assessinga co-substrate or a reaction product of adenosine conversion by anadenosine converting enzyme. In some embodiments, the adenosineconverting enzyme is an adenosine kinase and the reaction product isAdenosine 5′-monophosphate. In other embodiments, the adenosineconverting enzyme is an adenosine deaminase and the reaction productsare ammonium and inosine.

In one aspect, the present invention is directed to a method forassaying homocysteine (Hcy) in a sample, which method comprises: a)contacting a sample containing or suspected of containing Hcy with a Hcyco-substrate and a Hcy converting enzyme in a Hcy conversion reaction toform a Hcy conversion product and a Hcy co-substrate conversion product,wherein the Hcy co-substrate is S-adenosylmethionine (SAM), the Hcyconverting enzyme is S-adenosylmethionine (SAM)-dependent homocysteineS-methyltransferase, the Hcy conversion product is methionine (Met) andthe Hcy co-substrate conversion product is S-adenosyl-L-homocysteine(SAH), and b) assessing the SAH to determine the presence, absenceand/or amount of the Hcy in the sample, wherein the SAH is assessedwithout chromatographic separation.

The SAM can be used in any suitable form. For example, the SAM is addedto the sample directly. In another example, the SAM is produced by afurther reaction, e.g., produced from ATP and Met by a SAM synthase.

The SAH may be assessed by any suitable methods known in the art such asimmunological or enzymatic methods. For example, SAH may be assessed byassessing binding between SAH and mutant SAH binding enzyme, e.g., amutant SAH hydrolase that has binding affinity for Hcy, SAH or adenosinebut has attenuated catalytic activity. In one example, the assessment ofSAH does not involve an enzymatic reaction generating H₂O₂ and detectionof H₂O₂.

In another example, the SAH may be assessed by using an antibody whichspecifically binds to the SAH. The antibody may be monoclonal orpolyclonal. The antibody may also be bound to a carrier matrix. Anysuitable immunoassay formats, e.g., sandwich and competition assayformats, can be used.

The methods of the invention may be used for assaying homocysteine inany sample, including, but not limited to, a body fluid or a biologicaltissue. The body fluid may be selected from the group consisting ofurine, blood, plasma, serum, saliva, semen, stool, sputum, cerebralspinal fluid, tears, mucus and amniotic fluid. In some embodiments, thebody fluid is blood. In some embodiments, the blood sample is furtherseparated into a plasma or serum fraction.

In some embodiments, prior to or concurrently with the contact betweenthe sample and the Hcy co-substrate and the Hcy converting enzyme,oxidized or conjugated Hcy in the sample is converted into reduced Hcy.In some embodiments, the sample is subjected to treatment bydithiothreitol, tris(2-carboxyethyl)-phosphine hydrochloride (TCEP) orother reducing agent, in appropriate amounts to produce freehomocysteine in the sample.

The method of the invention may further comprise a step of removing thereducing agent used to convert oxidized or conjugated Hcy into reducedHcy prior to or concurrently with contacting the sample with the Hcyco-substrate and the Hcy converting enzyme. For example, the reducingagent can be removed by addition of N-ethylmaleimide or otherthio-reacting compounds.

Still another aspect of the present invention is directed to a kit fordetermining presence or concentration of homocysteine in a sample, whichkit comprises: a) a Hcy converting enzyme; b) a Hcy co-substrate; and c)a reagent for assessing Hcy co-substrate conversion product.

In some embodiments, the Hcy co-substrate is S-adenosylmethionine (SAM),the Hcy converting enzyme is a S-adenosylmethionine (SAM)-dependenthomocysteine S-methyltransferase, the Hcy co-substrate conversionproduct is S-adenosyl-L-homocysteine (SAH), and the reagent forassessing Hcy co-substrate conversion product is a reagent for assessingSAH. In some embodiments, the kit further comprises a reagent, e.g., aSAH hydrolase, to generate Hcy from SAM, which is cycled into the Hcyconversion reaction by the SAM-dependent homocysteineS-methyltransferase to form a Hcy co-substrate based enzyme cyclingreaction system, and adenosine (Ado).

The invention also provides a kit for assaying Hcy in a sample, whichkit comprises: a) a S-adenosylmethionine (SAM)-dependent homocysteineS-methyltransferase; b) S-adenosylmethionine (SAM) or ATP, Met and a SAMsynthase; c) a SAH hydrolase; and d) a reagent for assessing adenosine(Ado). The reagent for assessing Ado may be an adenosine convertingenzyme other than the SAH hydrolase, such as an adenosine kinase and anadenosine deaminase.

The invention also provides a kit for assaying Hcy in a sample, whichkit comprises: a) a S-adenosylmethionine (SAM)-dependent homocysteineS-methyltransferase; b) S-adenosylmethionine (SAM) or ATP, Met and a SAMsynthase; and c) a reagent for assessing SAH, wherein the kit does notcomprise an enzyme or a reagent for generating H₂O₂ and a reagent fordetecting H₂O₂.

In some embodiments, the kit of the invention further comprises areducing agent such as dithiothreitol (DTT) or TCEP.

The kit of the invention may be in any suitable packaging and may alsoinclude instructions for practicing methods described herein. The kitmay optionally include additional components such as buffers.

The assays described herein can be used for any suitable purposes, e.g.,prognostic, diagnostic, drug screening or treatment monitoring purposes.The assays readily can be automated. In addition, the assays can beadapted for use in point of care systems and in home test kits. Forexample, blood test point of care systems can be adapted for measuringhomocysteine levels using methods provided herein. Home test kits mayalso be adapted for use with the methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary assay method for homocysteine. Hcy:L-homocysteine; SAM: S-adenosylmethionine; HMTase: SAM-dependenthomocysteine S-methyltransferase; and SAHase: S-adenosyl-L-homocysteinehydrolase.

FIG. 2 depicts a serum homocysteine dose response curve obtained in theexperiment described in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “homocysteine (Hcy)” refers to a compound with thefollowing molecular formula: HSCH₂CH₂CH(NH₂)COOH. Biologically, Hcy isproduced by demethylation of methionine and is an intermediate in thebiosynthesis of cysteine from methionine. The term “Hcy” encompassesfree Hcy (in the reduced form) and conjugated Hcy (in the oxidizedform). Hcy can conjugate with proteins, peptides, itself or other thiolsthrough disulfide bond.

As used herein, “homocysteine (Hcy) conversion reaction” refers to areaction in which a compound reacts with a Hcy molecule during which achemical group (e.g., a methyl group) is transferred from the compoundto the Hcy molecule to form reaction products. The compound that reactswith the Hcy molecule and provides the chemical group is referred to as“homocysteine (Hcy) co-substrate.” The enzyme that catalyzes thereaction is referred to as “homocysteine (Hcy) converting enzyme.” Thereaction product that contains the whole or part of the original Hcymolecule is referred to as “homocysteine (Hcy) conversion product.” Thereaction product that does not contain any element from the original Hcymolecule is referred to as “Hcy co-substrate conversion product.”

As used herein, “SAM-dependent homocysteine S-methyltransferase” refersto an enzyme that catalyzes formation of methionine andS-adenosyl-L-homocysteine (SAH) from homocysteine andS-adenosylmethionine (SAM). It is intended to encompass SAM-dependenthomocysteine S-methyltransferase with conservative amino acidsubstitutions that do not substantially alter its activity.

As used herein, “SAH hydrolase” refers to an ubiquitous eukaryoticenzyme, which is also found in some prokaryotes, which catalyzeshydrolysis of SAH to adenosine (Ado) and Hcy. SAH hydrolase alsocatalyzes the formation of SAH from Ado and Hcy. The co-enzyme of SAHhydrolase is NAD⁺/NADH. SAH hydrolase may have several catalyticactivities. In the hydrolytic direction, the first step involvesoxidation of the 3′-hydroxyl group of SAH (3′-oxidative activity) byenzyme-bound NAD⁺ (E-NAD⁺), followed by β-elimination of L-Hcy to give3′-keto-4′,5′-didehydro-5′-deoxy-Ado. Michael addition of water to the5′-position to this tightly bound intermediate (5′-hydrolytic activity)affords 3′-keto-Ado, which is then reduced by enzyme-bound NADH (E-NADH)to Ado (3′-reduction activity). It is intended to encompass SAHhydrolase with conservative amino acid substitutions that do notsubstantially alter its activity.

As used herein the term “assessing” is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the amount or concentration of the analyte, e.g., homocysteineor Ado, present in the sample, and also of obtaining an index, ratio,percentage, visual or other value indicative of the level of analyte inthe sample. Assessment may be direct or indirect and the chemicalspecies actually detected need not of course be the analyte itself butmay for example be a derivative thereof or some further substance.

As used herein, “adenosine deaminase” refers to an enzyme that catalyzesthe deamination of adenosine to form inosine. It is intended toencompass adenosine deaminase with conservative amino acid substitutionsthat do not substantially alter its activity.

As used herein, “adenosine kinase” refers to an enzyme that catalyzesthe formation of adenosine 5′-monophosphate and ADP from adenosine andATP. It is intended to encompass adenosine kinase with conservativeamino acid substitutions that do not substantially alter its activity.

As used herein, “serum” refers to the fluid portion of the bloodobtained after removal of the fibrin clot and blood cells, distinguishedfrom the plasma in circulating blood.

As used herein, “plasma” refers to the fluid, noncellular portion of theblood, distinguished from the serum obtained after coagulation.

As used herein, “production by recombinant means” refers to productionmethods that use recombinant nucleic acid methods that rely on wellknown methods of molecular biology for expressing proteins encoded bycloned nucleic acids.

As used herein, “fluid” refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, “sample” refers to anything which may contain an analytefor which an analyte assay is desired. The sample may be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include urine, blood, plasma, serum, saliva, semen,stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid orthe like. Biological tissues are aggregates of cells, usually of aparticular kind together with their intercellular substance that formone of the structural materials of a human, animal, plant, bacterial,fungal or viral structure, including connective, epithelium, muscle andnerve tissues. Examples of biological tissues also include organs,tumors, lymph nodes, arteries and individual cell(s).

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from, e.g., infection or genetic defect, andcharacterized by identifiable symptoms.

As used herein, “antibody” includes not only intact polyclonal ormonoclonal antibodies, but also fragments thereof (such as Fab, Fab′,F(ab′)₂, Fv), single chain (ScFv), a diabody, a multi-specific antibodyformed from antibody fragments, mutants thereof, fusion proteinscomprising an antibody portion, and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity. An antibody includes an antibody of anyclass, such as IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class.

B. METHODS FOR ASSAYING HOMOCYSTEINE

The invention provides a method for assaying homocysteine (Hcy) in asample, which method comprises: a) contacting a sample containing orsuspected of containing Hcy with a Hcy co-substrate and a Hcy convertingenzyme in a Hcy conversion reaction to form a Hcy conversion product anda Hcy co-substrate conversion product; and b) assessing said Hcyco-substrate conversion product to determine the presence, absenceand/or amount of said Hcy in said sample.

In some embodiments, the Hcy co-substrate conversion product is assessedwithout chromatographic separation.

In some embodiments, the Hcy converting enzyme is a S-adenosylmethionine(SAM)-dependent homocysteine S-methyltransferase. When a SAM-dependenthomocysteine S-methyltransferase is used as the Hcy converting enzyme,the Hcy co-substrate is S-adenosylmethionine (SAM), the Hcy conversionproduct is methionine (Met) and the Hcy co-substrate conversion productis S-adenosyl-L-homocysteine (SAH), and the SAH is assessed to determinethe presence, absence and/or amount of the Hcy in the sample.

Any S-adenosylmethionine (SAM)-dependent homocysteineS-methyltransferase that transfers a methyl group from SAM to Hcy can beused. For example, S-adenosylmethionine: L-homocysteineS-methyltransferase described by Shapiro and Stanley K (Methods Enzymol.17 Pt.B, Sulfur Amino acids, pp. 400-405 (1971)) and Shapiro S K(Biochim. Biophys. Acta. 29:405-9 (1958)) can be used. The homocysteineS-methyltransferase (EC 2.1.1.10) encoded by the nucleic acid having thefollowing GenBank Accession No. AF297394 and the amino acid sequencehaving the following GenBank Accession Nos. AAG10301, CAA16035,NP_(—)856132, NP_(—)302039, CAD97346, T51939, T51941 and CAC30428 canalso be used. Preferably, the SAM-dependent homocysteineS-methyltransferase from Escherichia coli. (Thanbichler et al., J.Bacteriol., 181(2):662-5 (1999)) or S. cerevisiae (Shapiro et al., J.Biol. Chem., 239(5):1551-6 (1964) and Thomas et al., J Biol. Chem.,275(52):40718-24 (2000)) can be used.

The SAM can be used in any suitable form. For example, the SAM is addedto the sample directly. In another example, the SAM is produced by afurther reaction, e.g., produced from ATP and Met by a SAM synthase.

SAH may be assessed using any methods known in the art. For example, SAHmay be assessed by using an antibody which specifically binds to SAH.Antibodies may be polyclonal or monoclonal. Examples of antibodiesspecific to SAH are described in U.S. Pat. Nos. 5,631,127 and 6,063,581.Antibodies specific for SAH can also be generated using methods known inthe art, for example methods described in U.S. Pat. Nos. 5,631,127 and6,063,581.

Any immunological assays may be used for detecting SAH with the antibodyspecific to SAH, for example, competition or sandwich assays insolutions or on a solid support, precipitation/aggregation assays. Insome embodiments, the SAH is assessed by contacting the sample reactedwith SAM-dependent homocysteine S-methyltransferase in the presence ofSAM with an antibody specific to SAH and with a detectable hapten forthe antibody other than the SAH, and wherein determining the presence oramount of the SAH is effected indirectly by determining the presence oramount of the detectable hapten either bound or not bound to theantibody. In some embodiments, the antibody is bound to a carriermatrix.

SAH may also be assessed using a mutant SAH hydrolase having bindingaffinity for SAH but has attenuated catalytic activity. These mutant SAHhydrolases and assay methods using mutant SAH hydrolases are describedin U.S. Pat. No. 6,376,210 and WO 03/060478.

SAH may also be assessed by converting SAH to adenosine and Hcy by SAHhydrolase, and the adenosine generated is assessed. In some embodiments,the SAH is contacted with a SAH hydrolase to generate Hcy from SAM,which is cycled into the Hcy conversion reaction by the SAM-dependenthomocysteine S-methyltransferase to form a Hcy co-substrate based enzymecycling reaction system, and adenosine (Ado), which is assessed todetermine the presence, absence and/or amount of the Hcy in the sample.

In some embodiments, the present invention provides a method forassaying homocysteine in a sample, which method comprises: a)methylating homocysteine, if present in a sample, using a methyl donor,e.g., S-adenosylmethionine (SAM) and a SAM-dependent homocysteineS-methyltransferase to form methionine and S-adenosyl-L-homocysteine(SAH); b) releasing adenosine (Ado) from said formed SAH and generatinghomocysteine using an enzyme S-adenosyl-L-homocysteine hydrolase; and c)assessing said released Ado to determine presence and/or amount ofhomocysteine in said sample. The method can further include measuringthe released amounts of Ado over time. Preferably, steps a) and b) arecycled to release said Ado at rate that can be correlated to theconcentration of homocysteine in the sample. Also preferably, the rateof release of the Ado is correlated with standard homocysteine values ofconcentration. Any suitable methyl donors and methyltransferases can beused. For example, the methyl donor can be SAM and the methylationenzyme can be a SAM-dependent homocysteine methyltransferase.Preferably, the methyl donor SAM is provided in a concentration of atleast approximately 5 μM. The rate of Ado formation can be measuredusing any suitable methods. For example, the rate of Ado formation canbe measured enzymatically. Preferably, the rate of Ado formation ismeasured using Ado deaminase, glutamate dehydrogenase, purine nucleosidephosphorylase, xanthine oxidase, peroxidase, adenosine kinase, or acombination of any two or more of these enzymes. These assay methods arefurther described herein.

The SAM can be used in any suitable form. For example, the SAM is addedto the sample directly. In another example, the SAM is produced by afurther reaction, e.g., produced from ATP and Met by a SAM synthase.

Any SAH hydrolase can be used. For example, the nucleic acid moleculescontaining nucleotide sequences with the GenBank accession Nos.M61831-61832 can be used in obtaining nucleic acid encoding SAHhydrolase (See Coulter-Karis and Hershfield, Ann. Hum. Genet.,53(2):169-175 (1989)). Also preferably, the nucleic acid moleculecontaining the sequence of nucleotides or encoding the amino acidsdescribed in U.S. Pat. No. 5,854,023 can be used to obtain SAHhydrolase.

Various reducing reagents can be used (for example DTT, TCEP, cysteine,mercaptoethanol, dithioerythritol, sodium borohydride, etc.), howeverDTT is particularly suitable, e.g., at about 5 mM concentration. DTTshould itself be stored at low pH and thus the assay kit canconveniently include a solution of DTT at a low pH (e.g., about 3) butwith a low buffer capacity and a separate solution of SAH-hydrolase,which may be partially or totally inactive, at substantially neutral pHand preferably buffered. When these solutions are combined, the enzymeis reactivated at neutral pH. This combination can if desired take placein the presence of the test sample, or with the test sample addedshortly thereafter. The other reducing agents mentioned above maysimilarly be used for both SAH-hydrolase stabilization/activation. TCEPcan be stored at a neutral pH, which allows the enzymes to be includedin the same reagent with the reducing agent.

The Ado may be assessed by any suitable methods known in the art such asimmunological or enzymatic methods. Generally methods relying uponphotometric (e.g. colorimetric, spectrophotometric or fluorometric)detection and immunological methods may be used as these mayparticularly readily be adapted for use in clinical laboratories.Methods based on enzymatic reaction or reaction with mono- or polyclonalantibodies can also be used, as these are simple and quick and can berelatively inexpensive to perform. For example, the Ado may be assessedby monitoring the reaction with enzymes which convert it directly orindirectly to products which may be detected photometrically, e.g.,spectrophotometrically. Suitable enzymes, which should of course benon-reactive with the other substrates of the homocysteine convertingenzyme, particularly homocysteine, include adenosine deaminase (whichconverts adenosine to inosine) and adenosine kinase (which convertsadenosine and ATP to ADP and phosphorylated adenosine). Such enzymes mayfurther be combined with other enzymes which act to convert the productsformed to further detectable products.

Thus exemplary Ado detection schemes useful in the assay of theinvention include:adenosine→FPIA detection+fluorescein labelled adenosine   (1)adenosine→inosine+NH₃   (2)α-ketoglutarate+NH₃+NAD(P)H→L-glutamate+NAD(P)⁺  (2a)inosine→hypoxanthine   (2b1)hypoxanthine+O₂→Xanthine+H₂O₂   (2b2)Xanthine+O₂→uric acid+H₂O₂   (2b3)2H₂O₂+4-AA(aminoantipyrine)+TOOS(N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine)→QuinoneDye+4 H₂O   (2b4)Adenosine+ATP→Adenosine-5′-P+ADP   (3)Phosphoenolpyruvate+ADP+H⁺→pyruvate+ATP (3a)pyruvate+NADH→Lactate+NAD⁺  (3b)

In scheme (1), an immunoassay is conducted and the fluorescein labelledadenosine can be detected.

In scheme (2), the reaction is catalyzed by an adenosine deaminase andthe ammonia generated by the adenosine deaminase reaction may readily bedetected using known methods, e.g., colorimetric techniques. Thus forexample the ammonia generated in the sample may be reacted to formcolored products, the formation of which may be detectedspectraphotometrically.

In scheme (2a), the reaction is catalyzed by L-glutamate dehydrogenaseand the NAD(P)⁺ can be spectraphotometrically detected at 340 nm.

In scheme (2b1), the reaction is catalyzed by a purine nucleosidephosphorylase. In schemes (2b2) and (2b3), the reactions are catalyzedby a xanthine oxidase. In scheme (2b4), the reaction is catalyzed by aperoxidase. The inosine and uric acid have distinctive UV absorptionproperties and can thus be monitored spectraphotometrically, by kineticmeasurements. However the use of UV detection of uric acid or inosinehas certain limitations in that the sensitivity of the method is ratherpoor and it requires a UV-light source and a UV-transparent samplecontainer. It may thus be more convenient to rely upon colorimetricdetection of the Quinone dye at 550 nm.

Alternatively, in scheme (2b); the xanthine oxidase reaction lendsitself to detection using fluorogens or chromogens, e.g., red-oxindicators, by assessing the reduction/oxidation potential, or bymeasuring O₂ consumption, or more particularly H₂O₂ formation, forexample by the use of electronic sensors. Numerous red-ox indicators canbe used for this purpose, and a wide range of methods are described inthe literature for assaying H₂O₂ and O₂ in solution. Indeed, H₂O₂ isfrequently detected in clinical assays. Hydrogen peroxide, for example,can also be assessed using the non enzymatic chemiluminescent reactionsof peroxioxalate and the acridinium esters, the latter in aqueoussolution at neutral pH.

In scheme (3), the reaction is catalyzed by an adenosine kinase. Inscheme (3a), the reaction is catalyzed by a pyruvate kinase. In scheme(3b), the reaction is catalyzed by a lactate dehydrogenase. The NAD(P)⁺generated in scheme (3b) can be spectraphotometrically detected at 340nm.

Any adenosine deaminase can be used for scheme (2). For example, theadenosine deaminase from bovine spleen (Sigma-Aldrich catalog Nos.A5168, 6648 and 5043), from calf intestinal mucosa (Sigma-Aldrichcatalog Nos. 01898, A9876 and A1030) or human adenosine deaminase fromhuman erythrocytes (Sigma-Aldrich catalog No. BCR647) can be used. Inanother example, the adenosine deaminase encoded by the nucleic acidshaving the GenBank accession No. U76422 (Human, see also Lai, et al.,Mol. Cell. Biol., 17(5):2413-24 (1997)) can be used.

Any purine nucleoside phosphorylase can be used for scheme (2b). Forexample, the purine nucleoside phosphorylase encoded by the nucleicacids having the following GenBank accession Nos. can be used: U88529(E. coli); U24438 (E. coli, see also Cornell and Riscoe, Biochim.Biophys. Acta, 1396(1):8-14 (1998)); U83703 (H. pylori); and M30469 (E.coli).

Any xanthine oxidase can be used for scheme (2b). For example, thexanthine oxidase encoded by the nucleic acids having the followingGenBank accession Nos. can be used: AF080548 (Sinorhizobium meliloti);and U39487 (Human, see also Saksela and Raivio, Biochem. J, 315(1):235-9(1996)).

Any adenosine kinase can be used for scheme (3). For example, theadenosine kinase encoded by the nucleic acids having the followingGenBank accession Nos. can be used: NM_(—)006721 (Homo sapiens);NM_(—)001532 (Homo sapiens); NM_(—)001 123 (Homo sapiens); NM_(—)021129(Homo sapiens); and BC003568 (Homo sapiens). The adenosine kinasedisclosed in U.S. Pat. No. 5,861,294, McNally et al., Biochem. Biophys.Res. Commun. 231:645-650(1997), and Singh et al., Eur. J. Biochem.241:564-571 (1996) can also be used.

Any glutamate dehydrogenase can be used for scheme (2a). For example,the glutamate dehydrogenase (or glutamic acid dehydrogenase) disclosedin Perez-de la Mora et al., Anal. Biochem., 180(2):248-52 (1989) andGore, Int. J. Biochem., 13(8):879-86 (1981) an be used.

Any pyruvate kinase can be used for scheme (3a). For example, thepyruvate kinase from porcine (Sigma-Aldrich catalog No. K4388), Bacillusstearothermophilus (Sigma-Aldrich catalog No. P1903), chicken muscle(Sigma-Aldrich catalog No. P5788) and rabbit muscle (Sigma-Aldrichcatalog No. 83330) can be used.

Any lactate dehydrogenase can be used for scheme (3b). For example, thelactate dehydrogenase from Human (Sigma-Aldrich catalog No. BCR404),Lactobacillus leichmanii (Sigma-Aldrich catalog No. 61306),Lactobacillus sp (Sigma-Aldrich catalog No. 59023) and rabbit muscle(Sigma-Aldrich catalog No. 61311) can be used.

The methods described herein can be used to assay any sample, e.g., abody fluid or a biological tissue. Exemplary body fluids include urine,blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinalfluid, tears, mucus and amniotic fluid. Preferably, the body fluid to beassayed is blood. The blood sample can be assayed directly or be treatedbefore assaying. For example, the blood sample can be further separatedinto a plasma or serum fraction.

Prior to or concurrently with the contact between the sample and the Hcyco-substrate and the Hcy converting enzyme, oxidized or conjugated Hcyin the sample can be converted into reduced Hcy. In the plasma or urine,significant proportions of the homocysteine present may be bound bydisulphide linkage to circulating proteins, such as albumin, andhomocysteine may also be present in the form of other disulphidederivatives (generally homocysteine—cysteine conjugates). To obtain anestimate of total homocysteine present in the sample it may therefore bedesirable to treat the sample with a reducing agent to cleave thedisulphide bonds and liberate free homocysteine.

Any suitable reducing agent can be used. Disulphides are easily andspecifically reduced by thiols (e.g. tri-n-butylphosphine (TBP),dithiothreitol (DTT), dithioerythritol (DTE), 2-mercapto-ethanol,cysteine-thioglycolate, thioglycolic acid,tris(2-carboxyethyl)phosphine, free metals, glutathione and similarcompounds). Direct chemical reduction can be achieved using borohydrides(e.g. sodium borohydride) or amalgams (e.g. sodium amalgam) or morespecialized reagents such as phosphines or phosphorothioates can beused. Disulphide reduction is reviewed by Jocelyn in Methods ofEnzymology 143: 243-256 (1987) where a wide range of suitable reducingagents is listed. The reducing agent can also betris(2-carboxyethyl)-phosphine hydrochloride (TCEP). Preferably, thedithiothreitol or TCEP is provided in a concentration of up toapproximately 30 mM.

The method of the invention may further comprise a step of removing thereducing agent used to convert oxidized or conjugated Hcy into reducedHcy prior to or concurrently with contacting the sample with the Hcyco-substrate and the Hcy converting enzyme. For example, the reducingagent can be removed by addition of N-ethylmaleimide or otherthio-reacting compounds

C. KITS FOR ASSAYING HOMOCYSTEINE

In another aspect, the present invention is directed a kit for assayingHcy in a sample, which kit comprises: a) a Hcy converting enzyme; b) aHcy co-substrate; and c) a reagent for assessing Hcy co-substrateconversion product.

In some embodiments, the Hcy co-substrate is S-adenosylmethionine (SAM),the Hcy converting enzyme is a S-adenosylmethionine (SAM)-dependenthomocysteine S-methyltransferase, and the Hcy co-substrate conversionproduct is S-adenosyl-L-homocysteine (SAH). In some embodiments, thereagent for assessing Hcy co-substrate conversion product SAH is anantibody that specifically binds to SAH.

In another aspect, the invention is directed to a kit for assaying Hcyin a sample, which kit comprises: a) a S-adenosylmethionine(SAM)-dependent homocysteine S-methyltransferase; b)S-adenosylmethionine (SAM) or ATP, Met and a SAM synthase; c) a SAHhydrolase; and d) a reagent for assessing adenosine (Ado).

In still another aspect, the invention is directed to a kit for assayingHcy in a sample, which kit comprises: a) a S-adenosylmethionine(SAM)-dependent homocysteine S-methyltransferase; b)S-adenosylmethionine (SAM) or ATP, Met and a SAM synthase; and c) areagent for assessing SAH, wherein the kit does not comprise an enzymeor a reagent for generating H₂O₂ and a reagent for detecting H₂O₂.

In some embodiments, the reagent for assessing Ado comprises anadenosine converting enzyme other than the SAH hydrolase. In someembodiments, the adenosine converting enzyme is an adenosine kinase. Inother embodiments, the adenosine converting enzyme is an adenosinedeaminase.

The kit described herein can further comprise a reducing agent, e.g.,dithiothreitol or tris(2-carboxyethyl)-phosphine hydrochloride (TCEP).

The kits of the invention may be in any suitable packaging. For example,the packages discussed herein in relation to diagnostic systems arethose customarily utilized in diagnostic systems. Such packages includeglass and plastic, such as polyethylene, polypropylene andpolycarbonate, bottles and vials, plastic and plastic-foil laminatedenvelopes and the like. The packages may also include containersappropriate for use in auto analyzers. The packages typically includeinstructions for performing the assays described herein.

D. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 GLDH—NADH Coupling to Detect NH₄ ⁺ Generated by the EnzymaticCycling using Purified SAM

In this study, the following coupled enzymatic cycling reactions areused:

 Adenosine→Inosine+NH₄ ⁺  (3)NH₄ ⁺+α-Ketoglutarate+NAD(P)H→Glutamate+NAD(P)   (4)

In scheme (1), the reaction is catalyzed by a SAM-dependent homocysteineS-methyltransferase. In scheme (2), the reaction is catalyzed by a SAHhydrolase. In scheme (3), the reaction is catalyzed by an adenosinedeaminase. In scheme (4), the reaction is catalyzed by a L-glutamatedehydrogenase. The NAD(P)⁺ is spectraphotometrically detected at 340 nm.A more deteiled descirption of the reagents used in this study is setforth in the following Tables 1 and 2. TABLE 1 Compositions of Reagent 1Chemical Reagent 1 Concentration Potassium phosphate 15 mM NAD(P)H 5 mMGLDH 2 KU/L BSA 1.2 g/L Adenosine Deaminase 50 KU/L Homocysteinemethyltransferase 10 KU/L DTT 0.2 mM α-ketoglutarate 30 mM SAM 3 mM

TABLE 2 Compositions of Reagent 2 Chemicals Reagent 2 ConcentrationTris-HCl 15 mM BSA 1.2 g/L SAH hydrolase 10 KU/L

In this study, 180 μl of reagent 1 was mixed with 20 μl of a serum orplasma sample to be tested and the mixture was incubated at 37° C. for 5minutes. Sixty (60) μl of reagent 2 was then added to the mixture andwas incubated at 37° C. for another 5 minutes. The change of theabsorbance at 340 nm was measured for 2-5 minutes after the reagent 2was added. One exemplary test result was shown in FIG. 2.

Example 2 GLDH—NADH Coupling to Detect NH₄ ⁺ Generated by the EnzymaticCycling using SAM Concurrently Converted by SAM Synthase from ATP andMethionine

In this study, the following coupled enzymatic cycling reactions areused:

 Adenosine→Inosine+NH₄ ⁺  (4)NH₄ ⁺+α-Ketoglutarate+NAD(P)H→Glutamate+NAD(P)   (5)

In scheme (1), the reaction is catalyzed by a SAM Synthase. In scheme(2), the reaction is catalyzed by a SAM-dependent homocysteineS-methyltransferase. In scheme (3), the reaction is catalyzed by a SAHhydrolase. In scheme (4), the reaction is catalyzed by an adenosinedeaminase. In scheme (5), the reaction is catalyzed by a L-glutamatedehydrogenase. The NAD(P)⁺ is spectraphotometrically detected at 340 nm.A more deteiled descirption of the reagents used in this study is setforth in the following Tables 3 and 4. TABLE 3 Compositions of Reagent 3Chemical Reagent 3 Concentration Good's buffer 15 mM NAD(P)H 5 mM GLDH 2KU/L BSA 1.2 g/L TCEP 0.2 mM α-ketoglutarate 30 mM ATP 10 mM Methionine5 mM SAM Synthase 10 KU/L Adenosine Deaminase 50 KU/L Homocysteinemethyltransferase 20 KU/L ZnCl2 10 mM

TABLE 4 Compositions of Reagent 4 Chemicals Reagent 4 ConcentrationSodium phosphate 15 mM BSA 1.2 g/L SAH hydrolase 10 KU/L

In this study, 270 μl of reagent is mixed with 20 μl of a serum orplasma sample to be tested and the mixture is incubated at 37° C. for 5minutes. Sixty (90) μl of reagent 2 is then added to the mixture and wasincubated at 37° C. for another 5 minutes. The change of absorbance at340 nm is measured for 2-5 minutes after the reagent 2 is added.

Example 3 Adenosine Kinase-Pyruvate-Kinase-Lactate Dehydrogenase-NADHCoupling to Detect Adenosine Generated by the Enzymatic Cycling

In this study, the following coupled enzymatic cycling reactions areused:

 Adenosine+ATP→ADP+AMP   (3)ADP+PEP→Pyruvate+ATP   (4)Pyruvate+NADH→Lactate+NAD   (5)

In scheme (1), the reaction is catalyzed by a SAM-dependent homocysteineS-methyltransferase. In scheme (2), the reaction is catalyzed by a SAHhydrolase. In scheme (3), the reaction is catalyzed by an adenosinekinase. In scheme (4), the reaction is catalyzed by a pyruvate kinase.In scheme (5), the reaction is catalyzed by a lactate dehydrogenase. TheNAD(P)⁺ is spectraphotometrically detected at 340 nm. A more deteileddescription of the reagents used in this study is set forth in thefollowing Tables 5 and 6. TABLE 5 Compositions of Reagent 5 ChemicalReagent 5 Concentration Potassium phosphate 15 mM NADH 5 mM GLDH 2 KU/LBSA 1.2 g/L Adenosine Kinase 10 KU/L Homocysteine methyltransferase 10KU/L DTT 0.2 mM MgCl₂ 15 mM Pyruvate Kinase 5 KU/L Lactate Dehydrogenase25 KU/L SAM 3 mM

TABLE 6 Compositions of Reagent 6 Chemicals Reagent 6 ConcentrationTris-HCl 15 mM BSA 1.2 g/L SAH hydrolase 10 KU/L

In this study, 180 μl of reagent is mixed with 20 μl of a serum orplasma sample to be tested and the mixture is incubated at 37° C. for 5minutes. Sixty (60) μl of reagent 2 is then added to the mixture and wasincubated at 37° C. for another 5 minutes. The change of absorbance at340 nm is measured for 2-5 minutes after the reagent 2 is added.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

1-13. (canceled)
 14. A method for assaying homocysteine (Hcy) in asample, which method comprises: a) contacting a sample containing orsuspected of containing Hcy with a Hcy co-substrate and a Hcy convertingenzyme in a Hcy conversion reaction to form a Hcy conversion product anda Hcy co-substrate conversion product, wherein the Hcy co-substrate isS-adenosylmethionine (SAM), the Hcy converting enzyme is aS-adenosylmethionine (SAM)-dependent homocysteine S-methyltransferase,the Hcy conversion product is methionine (Met) and the Hcy co-substrateconversion product is S-adenosyl-L-homocysteine (SAH), b) assessing theHcy co-substrate conversion product SAH to determine the presence,absence and/or amount of the Hcy in the sample, wherein the SAH isassessed without chromatographic separation.
 15. The method of claim 14,wherein the assessment of SAH does not involve an enzymatic reactiongenerating H₂O₂ and detection of H₂O₂.
 16. The method of claim 14,wherein the SAM is added to the sample.
 17. The method of claim 14,wherein the SAM is produced from ATP and Met by a SAM synthase. 18-20.(canceled)
 21. A kit for assaying Hcy in a sample, which kit comprises:a) a S-adenosylmethionine (SAM)-dependent homocysteineS-methyltransferase; b) S-adenosylmethionine (SAM) or ATP, Met and a SAMsynthase; and c) a reagent for assessing SAH, wherein the kit does notcomprise an enzyme or a reagent for generating H₂O₂ and a reagent fordetecting H₂O₂.